Hybrid solid supports useful for oligonucleotide production

Abstract

A method for preparing a crosslinked polymer coated controlled porosity glass (CPG) particle is provided. The method involves mixing CPG particles in a solution comprising polyvinylbenzylchloride and a first solvent at a temperature below 10° C. A second solvent is added and a crosslinking agent is added to the mixture. The first solvent is removed rapidly within 1½ hours of addition of the crosslinking agent. The crosslinking reaction is permitted to proceed and the mixture is then cooled and treated to remove any remaining solvent. The resulting coated CPG particles are washed and dried. Also provided a polymer coated CPG particles using for loading ligand thereon.

The invention claimed is: 
     
       1. A method for preparing a conformal polymer-coated controlled porosity glass (CPG) particle useful for oligonucleotide production, said method being performed in the absence of a coupling agent to link the coating to the substrate of the CPG particle and comprising:
 (a) cooling CPG particles having pores with a mean average diameter of: 500 Angstroms to 4000 Angstroms (50 nm to 400 nm) to a temperature below about 10° C.; 
 (b) mixing the cooled CPG particles and a cold solution comprising a coating compound and a first solvent, wherein the coating compound is a polymer comprising monomeric subunits or a polymerizable monomer selected from the group consisting of a vinylbenzylchloride, an acrylic, a styrene, polymers thereof, and mixtures thereof; 
 (c) combining the CPG particles and the coating compound solution resulting from the mixture of (b) with a second solvent which differs from the first solvent and a crosslinking agent while maintaining the CPG particles at a temperature below about 10° C.; 
 (d) removing the first solvent from the coating compound solution while retaining the second solvent in the coating compound solution and maintaining the coating compound solution at a temperature below about 10° C.; 
 (e) heating the CPG mixture of (d) which comprises the second solvent to a temperature of about 38° C. to about 65° C. under an inert gas, to permit the crosslinking agent to react with the groups in the monomeric subunits or polymerizable monomers of the coating compound so as to provide the CPG particles with a conformal polymeric coating cross-linked in the range of about 2 to 40%, 
 wherein the crosslinked conformal polymeric coating layer is on the surface of the CPG particle and within its pores without changing the shape of the coated CPG particles, and whereby the conformal coated pores within the conformal coated CPG particles retain an average pore size of at least 90% of the average pore size of the uncoated CPG particles, based on dry pore size, wherein the second solvent has a boiling point higher than the reaction temperature of step (e); 
 (f) cooling the coated CPG particles; 
 (g) washing and drying the coated CPG particles, and 
 (h) derivatizing the conformal coated CPG particles with an amino functional group suitable for nucleoside loading. 
 
     
     
       2. The method according to  claim 1 , wherein in step (a) the CPG particles are cooled to a temperature in the range of about −20° C. to about 0° C. 
     
     
       3. The method according to  claim 1 , wherein the coating compound solution of (b) comprises the coating compound polyvinylbenzylchloride dissolved in dichloromethane. 
     
     
       4. The method according to  claim 3 , wherein the coating compound solution comprises about 400 uM of chloromethyl groups per gram of CPG. 
     
     
       5. The method according to  claim 1 , wherein the cross-linking compound is diaminobutane (DAB). 
     
     
       6. The method according to  claim 5 , wherein the DAB is in a solution comprising dichloromethane. 
     
     
       7. The method according to  claim 1 , wherein the first solvent is removed within 1½ hours of adding the crosslinking agent. 
     
     
       8. The method according to  claim 1 , wherein the CPG particles are heated to a temperature in the range of about 38 to about 65° C. under a nitrogen blanket. 
     
     
       9. The method according to  claim 8 , wherein the reaction is permitted to proceed for about 12 to 24 hours. 
     
     
       10. The method according to  claim 1 , wherein the cooled and coated CPG particles are dried under a stream of nitrogen. 
     
     
       11. The method according to  claim 1 , wherein the coated CPG particles are washed in acetone. 
     
     
       12. The method according to  claim 1 , wherein the washed coated CPG particles are dried in a dessicator. 
     
     
       13. The method according to  claim 1 , wherein in (c) the crosslinking agent reacts with a benzylchloride moiety of a vinylbenzylchloride polymer forming coating compound. 
     
     
       14. The method according to  claim 1 , characterized in that the polymer forming the coating is poly(vinylbenzylchloride) polymer crosslinked in the range of about 5 to about 30%. 
     
     
       15. The method according to  claim 1 , wherein the CPG particles are about 10 microns to about 500 microns in size. 
     
     
       16. The method according to  claim 1 , wherein the uncoated CPG particles have a mean average pore size of about 1000 Angstrom. 
     
     
       17. The method according to  claim 1 , wherein said conformal coating comprises a thickness of about 20 to 100 Angstroms. 
     
     
       18. The method according to  claim 1 , wherein said conformal coated pores within the coated particle have a mean average diameter of 500 to 2000 Angstroms. 
     
     
       19. The method according to  claim 1 , wherein said coating comprises about 5 to about 25 w/w % of the total weight of the coated particle. 
     
     
       20. The method according to  claim 19 , wherein said coating comprises about 5 to about 10% w/w of the total weight of the coated particle. 
     
     
       21. A method for preparing a cross-linked poly(vinylbenzylchloride)-coated controlled porosity glass (CPG) particle useful for oligonucleotide synthesis, said method being performed in the absence of a coupling agent to link the coating to the substrate of the CPG particle and comprising:
 (a) cooling CPG particles having pores of a mean average diameter of about 500 Angstrom to about 3000 Angstrom to a temperature below about 5° C.; 
 (b) mixing the cooled CPG particles with a cold solution comprising a coating compound comprising polyvinylbenzylchloride and a first solvent; 
 (c) adding a second solvent comprising 1,4-dioxane to the mixture of (b); 
 (d) adding a diaminobutane (DAB) crosslinking agent to the mixture of (c); 
 (e) removing the first solvent from the mixture of (d) while retaining the second solvent in the mixture; 
 (f) heating the CPG mixture of (e) to a temperature of about 38° C. to about 65° C., to permit the crosslinking agent to react with the coating compound so as to provide the CPG particles with a conformal polymeric coating cross-linked in the range of about 10 to about 20%, wherein the second solvent has a boiling point higher than that of the first solvent and the reaction temperature for cross-linking and the second solvent differs from the first solvent; 
 (g) cooling the CPG mixture of (f) and removing any remaining solvent; 
 (h) washing the conformal coated CPG particles, wherein said crosslinked conformal coating layer is located on the surface of the CPG and within its pores without changing the shape of the particles, and wherein the coated particles retain which have an average pore size of at least 90% of the average pore size of the uncoated CPG particles based on dry pore size and drying 
 wherein steps (b)-(e) are performed while maintaining the mixture containing the particles at a temperature below about 10° C.; and 
 (i) derivatizing the conformal coated CPG with an amino functional group suitable for nucleoside loading. 
 
     
     
       22. The method according to  claim 21 , wherein the second solvent is 1,4-dioxane.